Basal forebrain volume and metabolism in carriers of the Colombian mutation for autosomal dominant Alzheimer’s disease

We aimed to study atrophy and glucose metabolism of the cholinergic basal forebrain in non-demented mutation carriers for autosomal dominant Alzheimer's disease (ADAD). We determined the level of evidence for or against atrophy and impaired metabolism of the basal forebrain in 167 non-demented carriers of the Colombian PSEN1 E280A mutation and 75 age- and sex-matched non-mutation carriers of the same kindred using a Bayesian analysis framework. We analyzed baseline MRI, amyloid PET, and FDG-PET scans of the Alzheimer’s Prevention Initiative ADAD Colombia Trial. We found moderate evidence against an association of carrier status with basal forebrain volume (Bayes factor (BF10) = 0.182). We found moderate evidence against a difference of basal forebrain metabolism (BF10 = 0.167). There was only inconclusive evidence for an association between basal forebrain volume and delayed memory and attention (BF10 = 0.884 and 0.184, respectively), and between basal forebrain volume and global amyloid load (BF10 = 2.1). Our results distinguish PSEN1 E280A mutation carriers from sporadic AD cases in which cholinergic involvement of the basal forebrain is already detectable in the preclinical and prodromal stages. This indicates an important difference between ADAD and sporadic AD in terms of pathogenesis and potential treatment targets.


Basal forebrain volume and metabolism in carriers of the Colombian mutation for autosomal dominant Alzheimer's disease
Stefan Teipel 1,2* , Alice Grazia 1 , Martin Dyrba 1 , Michel J. Grothe 3 & Nunzio Pomara 4 We aimed to study atrophy and glucose metabolism of the cholinergic basal forebrain in nondemented mutation carriers for autosomal dominant Alzheimer's disease (ADAD).We determined the level of evidence for or against atrophy and impaired metabolism of the basal forebrain in 167 non-demented carriers of the Colombian PSEN1 E280A mutation and 75 age-and sex-matched nonmutation carriers of the same kindred using a Bayesian analysis framework.We analyzed baseline MRI, amyloid PET, and FDG-PET scans of the Alzheimer's Prevention Initiative ADAD Colombia Trial.We found moderate evidence against an association of carrier status with basal forebrain volume (Bayes factor (BF 10 ) = 0.182).We found moderate evidence against a difference of basal forebrain metabolism (BF 10 = 0.167).There was only inconclusive evidence for an association between basal forebrain volume and delayed memory and attention (BF 10 = 0.884 and 0.184, respectively), and between basal forebrain volume and global amyloid load (BF 10 = 2.1).Our results distinguish PSEN1 E280A mutation carriers from sporadic AD cases in which cholinergic involvement of the basal forebrain is already detectable in the preclinical and prodromal stages.This indicates an important difference between ADAD and sporadic AD in terms of pathogenesis and potential treatment targets.
Pathological evidence suggests a cholinergic deficit in dementia stages of sporadic Alzheimer's disease (AD), characterized by reduced choline-acetyl transferase and acetylcholinesterase activity in cortical target regions of cholinergic projections from the basal forebrain, particularly the Nucleus basalis Meynert (NbM), and loss of cholinergic neurons in the NbM 1 .In early stages of sporadic AD, i.e. in people with an antemortem diagnosis of mild cognitive impairment (MCI), cholinergic basal forebrain neurons exhibited neurofibrillary tangles and cell shrinkage associated with accumulation of cortical amyloid 2,3 , but not yet frank neuron loss 4 .
Consistently, volumetric MRI studies demonstrated atrophy of the basal forebrain in sporadic MCI cases [5][6][7][8][9] and amyloid positive cognitively normal people [10][11][12] .FDG-PET studies found increased basal forebrain metabolic rate in sporadic MCI cases compared to normal controls 13,14 , which may indicate compensatory upregulation of regional metabolism in early stages of neurodegeneration or loss of cortical GABAergic inhibitory neurons.In contrast to sporadic AD, basal forebrain has not yet been studied in humans with autosomal dominant AD (ADAD).
Here, we studied baseline data from participants of the API Colombian trial recruited from a kindred harboring the Colombian NM_000021:c.839A>C,p.(Glu280Ala) (commonly known as PSEN1 E280A) mutation 15 , which is associated with early onset ADAD 16 .We determined volume and metabolism of the cholinergic basal forebrain in association with mutation carrier status and levels of cerebral amyloid.We hypothesized that nondemented PSEN1 E280A mutation carriers would exhibit atrophy and increased glucose metabolism of the basal forebrain compared with non-carriers, suggesting an early cholinergic deficit and compensatory hyperactivity.For comparison, we examined hippocampal atrophy, as some 17,18 , but not all 19,20 studies have shown hippocampal

Association of carrier status and brain volumes with cognitive scores
Within the CDR 0 cases, we found extreme evidence for an association of carrier status with delayed recall performance (RBANS delayed memory) (BF 10 = 12,062.2),indicating poorer performance in mutation carriers, controlling for age group, education, and sex.Evidence was inconclusive for attention (BF 10 = 0.454).Details are shown in Fig. 2.
Both in CDR 0 and in all cases combined, we found evidence against an association of normalized basal forebrain and hippocampus volumes with delayed recall.Evidence was in favor of an association of thalamus volume with delayed recall (Fig. 3), both in CDR 0 and in all cases combined, but not with attention, after controlling for age, sex, education, and carrier status.Details are shown in Table 2. Numbers in the CDR 0.5 subgroup were too small to run meaningful regression analyses separately in this group.

Voxel based analysis of MRI data
We found small areas of reduced volumes in PSEN1 E280A carriers compared with non-carriers in right predominant medial thalamus, consistent with the region of interest based analyses (Fig. 4), as well as small areas of increased brain volumes in carriers vs. non-carriers in bilateral fusiform gyrus, middle temporal gyrus and cerebellum.
Table 1.Demographic characteristics. 1 Bayesian contingency table: Bayes factor shows moderate evidence for no difference in proportion of sex between groups (BF 10 = 0.251). 2 Bayesian contingency table: Bayes factor shows extreme evidence for a difference in proportion of age-groups between groups (BF 10 = 3.8 × 10 6 ), with higher age in the noncarriers. 3Bayesian contingency table: Bayes factor shows moderate evidence for no difference in proportion of CDR global scores between groups (BF 10 = 0.138). 4Bayesian t-test: Bayes factor shows inconclusive evidence for a difference between groups (BF 10 = 1.384). 5Bayesian t-test: Bayes factor shows moderate evidence for no difference between groups (BF 10 = 0.168).www.nature.com/scientificreports/

Association of carrier status with regional metabolism
In an ANCOVA model, evidence was in favor of no difference between mutation carriers and non-carriers in pons normalized basal forebrain signal (BF 10 = 0.167), controlling for age, sex, and CDR score, suggesting a relatively preserved basal forebrain metabolism in mutation carriers (Fig. 5).We found anecdotal evidence against a difference of hippocampus metabolism (BF 10 = 0.582) and moderate evidence against a difference of thalamus metabolism (BF 10 = 0.165) in mutation carriers compared with non-carriers.

Voxel based analysis of FDG-PET data
We found reduced globally-normalized metabolism in bilateral superior parietal and posterior cingulate cortex, and increased metabolism in cerebellum and basal forebrain regions in mutation carriers (Fig. 6).

Amyloid sensitive AV45-PET data across groups
K-means clustering with R command kmeans resulted in two clusters with a threshold of approximately > 1.12 for amyloid positivity (Supplementary Fig. 1).We found extreme evidence in favor of an effect of carrier status on amyloid positivity (contingency

Association of volumes with global AV45-PET signal
We found inconclusive evidence for an association of global AV45-PET signal with normalized basal forebrain (BF 10 = 2.1) or hippocampus volume (BF 10 = 1.4), but very strong evidence in favor of an association of global AV45-PET signal with thalamus volume (BF 10 = 99.0).More amyloid signal was associated with smaller thalamus volume, after controlling for mutation carrier status.Details can be found in Supplementary Fig. 2. The effect of mutation carrier status on thalamus volume was fully mediated by the global AV45-PET signal, accounting for 87% of the covariance (Supplementary Fig. 3).

Discussion
Contrary to our primary hypothesis, we found evidence against an association of mutation carrier status with basal forebrain volume.In sporadic AD, atrophy of cholinergic basal forebrain is an early event and detectable in prodromal and even in preclinical stages of the disease [5][6][7][8][9] .Here, we found evidence against atrophy of the basal forebrain in preclinical and prodromal ADAD related to a PSEN1 E280A mutation.Basal forebrain atrophy has not been studied before in ADAD and also pathological studies have not investigated involvement of cholinergic basal forebrain neurons in ADAD cases.A cortical nicotinergic receptor deficit has been described in Swedish mutation carriers, however, this was based on autopsy in advanced stages of AD 31 .Preclinical studies demonstrated pathology of the cholinergic basal forebrain in transgenic mice with double or single amyloid precursor protein (APP) and presenilin1 mutations [32][33][34][35] .Specifically, in addition to confirming the presence of amyloid plaques, these studies showed a significant decrease in hippocampal choline acetyltransferase (ChAT) activity 32 , metabolic changes and DNA damage in basal forebrain 35 , no evidence of tau pathology but extensive inflammatory glial responses and increased trophic effects 34 .In contrast, one study analyzing ChAT activity, ChAT  www.nature.com/scientificreports/mRNA level, cholinergic neuron number and receptor binding in mutant PSEN1, APP and PSEN1/APP mice showed evidence of intact basal cholinergic innervation, even in the presence of extensive amyloid pathology 36 .
Our results raise the question whether these findings are transferable to humans with such mutations.Similar to the basal forebrain, we found evidence against atrophy of the hippocampus in the PSEN1 E280A mutation carriers.This finding in the relatively large cohort agrees with previous studies on subsamples of less than 30 asymptomatic carriers and 30 noncarriers each of the Colombian kindred that revealed no statistically significant reductions in hippocampal volume 19,20,24 .Likewise, a study in ADAD mutations other than PSEN1 E280A found no hippocampus atrophy in nine asymptomatic mutation carriers compared with nine control subjects, and even when 12 MCI mutation carriers were considered, the hippocampus was spared atrophy 21 .In carriers of different APP, PSEN1, and PSEN2 mutations, hippocampal atrophy was found from approximately 5 years before 17 to 8 years after the expected onset of the disease 37 , indicating a large variation of hippocampus atrophy trajectories across different mutations.
Interestingly, children and adolescents with the Colombian PSEN1 E280A mutation at age 9-17 years showed increased hippocampus, parahippocampus, and parietal and temporal lobe volumes compared with non-carriers 38 .Similarly, a study of individuals with different PSEN1 mutations found a significant increase of cortical thickness in parietotemporal regions in six asymptomatic mutation carriers on average 9.9 years before expected age of onset 39 .This is also consistent with a study in seven PSEN1 mutation carriers, distinct from the Colombian kindred, who showed accelerated hippocampus atrophy with transition to a symptomatic stage, but did not differ from controls in hippocampus volumes before this transition 18 .Similarly, in a cross-sectional analysis, cortical thickness in PSEN1 E280A carriers was higher in children and adolescent mutation carriers compared to agematched noncarriers 40 .The underlying mechanisms of the volume increases in young asymptomatic individuals with PSEN1 mutations are currently unresolved.The PSEN1 E280A mutation may be associated with early developmental changes or neuroinflammation with glial activation or neuronal hypertrophy 41 in response to neurotoxic amyloid and lead to early apparent increase of volume in AD vulnerable regions, only later followed by neurodegeneration and related brain atrophy.This also fits with the observation from mutation carriers of the DIAN cohort that did not show differences to non-carriers in hippocampus volume at an asymptomatic baseline, but mild rates of hippocampus atrophy were followed by a strong increase of rates of volume loss only after symptom onset 42,43 .The lack of hippocampal atrophy in our analysis may therefore reflect the asymptomatic to early symptomatic stage of PSEN1 E280A mutation carriers, in which there may be a transition from a developmental increase to a neurodegenerative decrease in hippocampal volume.Several studies, mostly using voxel-based analyses, reported atrophy of the thalamus in asymptomatic mutation carriers [21][22][23] , including individuals of the Colombian kindred 24 , reviewed in 44 .Early atrophy of the thalamus in ADAD is consistent with the early deposition of amyloid in this region and the striatum as shown using amyloid sensitive PIB-PET imaging in mutation carriers from the DIAN study 22 .Here, we found that atrophy of the thalamus, but not the basal forebrain or hippocampus 20 , was associated with a decrease in delayed memory performance in mutation carriers, both when considering all cases and when considering only asymptomatic cases.One study in a small sample of the Colombian kindred did not find a difference in whole thalamus volume and thalamic subregion volumes between mutation carriers and non-carriers, despite numerically larger volumes in the noncarriers 45 .
Consistent with our primary hypothesis, we found evidence for a preserved metabolism of basal forebrain in mutation carriers, consistent with previous studies in sporadic prodromal AD 13,14 .Regions with relatively preserved metabolism included basal forebrain, thalamus and hippocampus in the voxel based analysis.In contrast, other PSEN1 mutations showed pronounced hypometabolism of hippocampus in asymptomatic stages 46 , however, basal forebrain metabolism had not been assessed before.Hypometabolism in our analysis was mainly detected in superior parietal cortex, precuneus, and posterior cingulate gyrus, resembling the topography of cortical thickness reductions in the DIAN cohort asymptomatic mutation carriers 47 and the typical pattern of hypometabolism found in sporadic cases with amnestic or amyloid positive MCI 48 .
Amyloid load was much more pronounced in the mutation carriers than the non-carriers, consistent with the presence of a PSEN1 mutation and the young age of the cohort that implied a low risk for cerebral amyloidosis in non-mutation carriers.Different to previous analyses in sporadic prodromal and preclinical AD cases [10][11][12] , we only found anecdotal evidence for an association of global amyloid load with basal forebrain volume.In contrast, evidence was very strong for an association of global amyloid load with thalamus volume, and amyloid load fully mediated the association of mutation carrier status with thalamus volume.This is consistent with the pathogenic role of the PSEN1 E280A mutation leading to early-onset cerebral amyloidosis as the main determinant of subsequent neurodegeneration and cognitive decline 49 .

Limitations
We note the following limitations of this study: we lacked longitudinal follow-up since the study participants were included in the ongoing API ADAD Colombia trial so that access to longitudinal data was not possible.www.nature.com/scientificreports/ We also lacked information on the expected age of onset for the asymptomatic mutation carriers, which would have been very interesting to study in association with brain volumes and metabolism.The homogeneity of the genetic background is both an advantage and a disadvantage.It shows that the pathomechanistic effects of the PSEN1 E280A mutation lead to early involvement of thalamus but not of basal forebrain or hippocampus.It needs, however, to be shown if these results generalize to other presenilin1 mutations and even to presenilin2 and APP mutations.Basal forebrain volume and metabolism are surrogate markers for the integrity of the cholinergic basal forebrain, but not a direct estimate of neuronal structural and functional integrity 3 .However, the methods applied here match similar studies in sporadic AD allowing a direct comparison.

Conclusions
In this study, basal forebrain volume was not decreased in non-demented mutation carriers for ADAD and basal forebrain metabolism was relatively preserved.Early changes in delayed memory were associated with thalamus but not basal forebrain and hippocampus volume pointing to a different involvement of subcortical brain regions and relative sparing of cholinergic projection sites in prodromal ADAD compared with sporadic AD.This study highlights the importance of alternative disease mechanisms in ADAD and sporadic AD, at least for the PSEN1 E280A mutation, that may be relevant to understand different pathogenic roles of amyloid pathology for regional brain atrophy and metabolic changes in early stages of ADAD and sporadic AD.These findings may also have implications for considering initiation of cholinergic treatment not only in prodromal sporadic AD, but also in prodromal cases of ADAD.

Data source
Data were made available from the Alzheimer's Prevention Initiative (API) Autosomal-Dominant Alzheimer's Disease Colombia Trial (NCT01998841) baseline data 50 .The trial design has been described before 51 .Originally, 252 participants had been enrolled, however, data on 10 participants had to be excluded already from the trial baseline data presentation 52 to protect their confidentiality, genetic status, and trial integrity.Informed consent was obtained from all participants and study partners before any study related procedures were conducted.The trial was approved by the Colombian Health Authority (Instituto Nacional de Vigilancia de Medicamento).All consent procedures were conducted in accordance with international and local ethics committee standards and after ethics committee approval.All research was performed in accordance with relevant guidelines and regulations, including the Helsinki Declaration of 1975 and its later amendments.Adherence to STROBE reporting guidelines is documented in Supplementary Table 2.

Participants
The API Colombia trial included individuals who carry the PSEN1 E280A autosomal-dominant mutation 15 , do not meet criteria for MCI 53 or dementia due to AD 54 , and are between ≥ 30 years and ≤ 60 years of age 51 .In addition, age-and sex matched members of the same kindred without the mutation were included.Detailed inclusion and exclusion criteria can be found in 51 .

Cognitive measures
Measures included Clinical Dementia Rating (CDR) global score 55 which we used for stratifying participants in unimpaired (CDR = 0) and slightly impaired (CDR = 0.5) samples.In addition, we used the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) Memory and attention scores 56 .
FDG PET data acquisition FDG-PET images were acquired on a Siemens/CTI Biograph PET/CT system, using intravenous administration of 5 mCi (185 million Bq) of FDG after a 30-min radiotracer uptake period when resting with open eyes in a darkened room, followed by a 30-min dynamic emission scan (six 5-min frames).Images were reconstructed with computed tomographic attenuation correction.

Amyloid PET data acquisition
Florbetapir scans were acquired on the same Siemens Biograph PET/CT system as the FDG-PET data, using an intravenous bolus injection of ~ 11 mCi (9.3-14.7 mCi) of florbetapir, a CT scan for correction of radiation attenuation, a 50-min radiotracer uptake-period, and a 20-min dynamic emission scan in four frames (4 × 300 s) 57 .PET images were reconstructed using an ordered subset expectation maximization (OSEM) algorithm and attenuation-corrected, frames were evaluated for adequate count statistics and absence of head motion 57 .

Image data processing
MRI data MRI data were processed by using statistical parametric mapping (SPM12, Wellcome Trust Center for Neuroimaging) and the CAT12.3-toolbox(http:// dbm.neuro.uni-jena.de/ cat) implemented in MATLAB R2019 (Math-Works, Natick, MA).First, MRI scans were automatically segmented into grey matter (GM), white matter (WM) and cerebrospinal fluid (CSF) partitions of 1.5 mm isotropic voxel-size using the prior free Adaptive Maximum A Posterior (AMAP) segmentation routine of the CAT12-toolbox.The resulting GM and WM partitions of each subject in native space were then high-dimensionally registered to the MNI reference template using the DARTEL algorithm 58 .Individual flow-fields resulting from the DARTEL registration to the reference template were used to warp the GM segments and voxel-values were modulated for volumetric changes introduced by the highdimensional normalization, such that the total amount of GM volume present before warping was preserved.For extraction of basal forebrain volume, we used a cytoarchitectonic map of BF cholinergic nuclei in MNI space, derived from combined histology and MRI of a post-mortem brain, as described previously 59 .Hippocampus and thalamus volumes were derived using the Hammers brain atlas regions of interest 60 .

PET data
Images were preprocessed using SPM12 implemented in Matlab 2019 61 .First, each subject's averaged PET frames were co-registered to their corresponding T1-weighted MRI scan.Then, the coregistered PET images were spatially normalized to the MNI reference template using the deformation parameters derived from the normalization of their corresponding MRI scans.
For extraction of Florbetapir SUVR we used the Centiloid cortical mask and normalized the PET signal to the whole cerebellum Centiloid mask 62 .For FDG-PET we extracted regional SUVR values using the basal forebrain region and Hammers brain atlas regions of interest for hippocampus and thalamus 60 , and normalized the PET signal to the signal of the pons.

Statistical analysis
For region of interest based analyses we used Bayesian ANCOVA with Bayes factor (BF) hypothesis testing with volume or metabolism as dependent variable, mutation carrier status as independent variable, and age, sex, CDR

Figure 1 .
Figure 1.Association of carrier status with brain volumes.Boxplots and violin plots featuring volumes of basal forebrain, hippocampus, and thalamus according to mutation carrier status.Each volumetric measure was normalized to total intracranial volume (TIV).

Figure 2 .
Figure 2. Association of carrier status with cognitive scores.Boxplots and violin plots featuring distribution of cognitive scores of delayed recall memory and attention according to mutation carrier status.

Figure 3 .
Figure 3. Association of thalamus volume with delayed recall across.Scatter plot of delayed recall regressed on thalamus volume (normalized to total intracranial volume) split according to mutation carrier status.

Figure 4 .
Figure 4. Association of carrier status with voxel-wide grey matter volume.Relative increase of signal in mutation carriers vs. non-carriers (green) and relative decrease in mutation carriers (red).Cluster of at least 50 voxels with p < 0.001.Blue figures on top of each scan indicate the z-coordinate in MNI space, corresponding to the level of the axial sections.Right of image is right of brain (view from superior).

Figure 5 .
Figure 5. Association of carrier status with basal forebrain FDG-PET signal.Boxplot and violin plot featuring distribution of FDG-PET signal of the basal forebrain normalized to pons signal, according to mutation carrier status.

Figure 6 .
Figure 6.Voxel-wise association of carrier status with FDG-PET signal.(a) Effects projected on the rendered surface of an MRI scan in MNI space.Relative increase of signal in mutation carriers vs. non-carriers (green) and relative decrease in mutation carriers (red).Cluster of at least 50 voxels with p < 0.001.(b) Projection on coronal brain slices.Relative increase of signal in mutation carriers vs. non-carriers (green) and relative decrease in mutation carriers (red).Cluster of at least 50 voxels with p < 0.001.Blue figures on top of each coronal scan indicate the y-coordinate in MNI space, corresponding to the level of the coronal sections.Right of image is right of brain (view from posterior).

Table 2 .
Association of brain volumes with cognitive scores.β-unstandardized parameter estimates for the association between volume and cognitive function from the ANCOVA model, controlling for age, sex, education, CDR score (in CRD 0 + 0.5 cases), and mutation carrier status.